1. Field of the Invention
The invention generally relates to the prevention and treatment of cyclic GMP-dependent pathophysiologies and to the development of drugs for use therein. More particularly, the invention pertains to such prevention, treatment and drug development using methods and compositions that employ a heme-deficient αβCys105 mutant soluble guanylyl cyclase (sGC) enzyme or gene.
2. Description of Related Art
Soluble guanylyl cyclase (sGC), is a crucial enzyme in the NO/cGMP dependent pathway. sGC-mediate increase in intracellular cGMP affects smooth muscle relaxation, platelet aggregation, leukocyte adhesion, cell proliferation and migration, neurotransmission and other effects (1). sGC is a heterodimeric protein composed of one α and one β subunit (2). Two isoforms for each subunit ((α1, α2, β1, β2) have been described, with the heterodimer α1β1 ubiquitously expressed. Both subunits are required for a fully active sGC (3). Deletion analysis of the α and β subunits (4) and subsequent site-directed mutagenesis studies delineated some aspects of the domain structure of sGC. The NH2-terminal domains of both subunits play a crucial role in the mediation of the NO-dependent activation of sGC (4). The C-terminal portion of each subunit contains a cyclase homology domain found in all nucleotide cyclases. The C-terminal domains of both subunits are required for the assembly of an active catalytic center (5). The sGC heterodimer contains a heme prosthetic group (6). The heme-containing N-terminal domain, also referred to as the regulatory domain, is separated from the C-terminal catalytic domain by a region believed to be important for the dimerization of sGC (7), although the role of this domain in dimerization remains to be tested. Soluble guanylyl cyclases are often referred to as Nitric Oxide (NO) receptors. NO binds to the heme moiety of sGC (8) and coincide with stimulation of sGC (9), which reaches several hundred-fold with purified enzyme (10–12). Extensive analysis of UV-Vis (11,13), EPR (14) and Raman (15,16) spectra described the transformation of the sGC heme prosthetic group upon binding of nitric oxide. In the absence of NO sGC heme is 5-coordinated with a histidine 105 residue of the β subunit as the axial ligand. Binding of NO to Fe++ and formation of nitrosyl heme result in the disruption of the histidine-heme coordinating bond and displacement of iron from the protoporphyrin plane. The kinetic and mechanistic aspects of these transformations are the subject of considerable investigation (17–20). Site-directed mutagenesis studies identified His105 of the β subunit as the axial ligand for the heme moiety (21, 22) and the first 365 residues of the β subunit are sufficient to bind and retain the heme group (23). Although the connection between the formation of the nitrosyl heme and activation of cGMP synthesis is well documented and accepted, little is known about the coupling mechanism between these two events. While the perturbations in the heme moiety of sGC upon NO-binding are well described, the mechanism by which the heme-containing domain regulates sGC catalytic center is not understood. One of the most commonly accepted hypotheses proposes that NO-induced release of the His105 residue allows His105 to exert its function and stimulate the enzyme (24, 25). Toward elucidating the main mechanism of physiological activation and pharmacological inhibition of cGC, there are reports describing the substitution of the heme-coordinating Histidine 105 residue of the bovine β subunit with phenylalanine (21, 40). As this substitution resulted in elimination of NO-dependent regulation of sGC, it proved the crucial role of βHis105 residue in activation of sGC enzyme. To analyze the role of the heme moiety in the activation of sGC by YC-1, the heme-coordinating histidine was substituted with a cysteine residue (26), which is known to coordinate heme in other enzymes, e.g. nitric oxide synthase (43). This report demonstrated that His105 residue is important for the activation of sGC through allosteric activator YC-1, but YC-1-dependent activation can occur without heme, albeit less efficiently.
In summary, it is known that soluble guanylyl cyclase (sGC) is an important enzyme that is involved in the regulation of cardiovascular homeostasis and pathologies (blood pressure, atherosclerosis, septic shock), neurotransmission and sensory perception. This enzyme is the target of a group of compounds known as NO-donors, or such known and widely used drugs as nitroglycerin. Upon activation by these drugs the enzyme synthesizes intracellular messenger cGMP and regulates a number of cellular processes. The enzyme acts as a heterodimer whose activity is regulated by the ferrous heme moiety. The sGC activation methodologies presently in use or in development rely on activation of sGC enzyme by delivering pharmacological compounds (mainly NO donors or allosteric regulators of sGC) or on delivery of NOS genes coding for enzymes that produce NO in order to activate sGC. There is no currently available method that can increase and sustain cGMP levels without drug administration. Another drawback of the methods currently used in practice today is that the effects of nitroglycerin and NO-donors are transient and the patients taking it often develop tolerance to their effects. In addition, some of the allosteric regulators have toxic effects on cellular level.
Inhibition of sGC activity has been shown to be effective in the prevention of septic shock in animals. Conventional inhibitors of sGC are based on the oxidation of the heme moiety of the enzyme. Those inhibitors are not specific since they also affect other heme-containing enzymes. The conventional inhibitors are also not very effective due to the large excess of hemoglobin and myoglobin proteins which buffer the effect of the inhibitors.